Hepatitis B virus (HBV) DNA contains several open reading frames, one of which is the env gene. This gene codes for 3 closely related proteins; preS1+preS2+S, preS2+S and S, in their respective 5'-3' genetic order and which comprise the structural envelope, or surface ("S") proteins. Collectively, the PreS1+PreS2+S, PreS2+S, and S proteins are referred to as hepatitis B virus surface proteins. The preS2+S and S proteins are capable of assembling into a structure known as the 22 nanometer (22 nm) particle or Australia antigen. The 22 nm particles can consist of a heterogeneous combination of S proteins or homogeneously of one form of S protein. All of the S related proteins are found in the intact HBV virion.
Through the use of recombinant DNA technology it has been demonstrated that the DNA coding for the S proteins can be introduced into various host cells (e.g. E. Coli, yeast, insect and mammalian cell cultures) resulting in the synthesis of preS1+preS2+S, preS2+S and S proteins, and the subsequent formation of 22 nm particles from preS2+S and S proteins. All three forms of the S protein are known to be immunogenic in vivo and antibodies to the S proteins are protective, with the preS2+S protein being immunodominant by virtue of the preS2 region. The preS2 region may function as a cellular membrane interaction sequence during the course of virus replication.
Expression of the preS2+S protein in yeast cells has demonstrated that the preS2+S protein interacts with yeast cell membranes and that purification of the preS2+S protein can be facilitated by this property.
Currently, a method exists for the purification of substantially pure membrane bound preS2+S protein. This method has several drawbacks which include: a) substantial amounts of contaminating yeast proteins at early stages of the purification scheme, b) proteolytic degradation of the preS2+S protein due to high levels of contaminating yeast proteases, c) the addition (and subsequent removal) of protease inhibitors to combat proteolytic degradation of preS2+S protein and, d) product yield reduction due to the culmination of the above factors. In addition, this protein, so purified, is being used as a vaccine in humans for the prevention of HBV infection.
It cannot be predicted what methods of purification will be useful for recombinant proteins since recombinant proteins are presented in a form which is usually atypical of the natural or classical form. For this reason, recombinant proteins frequently require novel combinations of known procedures or entirely new methods of purification.
In addition, vaccine preparations for human use require extreme purity which introduces even greater unpredictability in the outcome of a purification scheme.